This project will investigate the mechanism of the active transport system, or pump, for sodium and potassium ions across animal cell membranes. Ion translocation is coupled to phosphorylation of sodium, potassium transport adenosine triphosphatase, (Na,K)ATPase, from ATP with release of ADP and inorganic phosphate. The phosphate group of the phosphoenzyme is a most sensitive and available reporter group of the reactive state of the enzyme. The project will investigate the transient kinetics of its formation and breakdown under the influence of ligands of the enzyme, such as ATP, ADP, Pi, Na, K, Mg2 ions, the specific inhibitor ouabain, and their congeners. Phosphorylation will be estimated from 32P ATP, 32Pi or both (in different samples). The approach will be to isolate partial reactions and reactive states of the enzyme bounded by slow steps in the reaction pathway. Preference will be given to studies of interactions of ligand binding at equilibrium. The enzyme will be obtained principally in membrane preparations from kidney by the method of Jorgensen with improvements to increase the specific activity. The project will test the extent to which the phosphoenzyme can be resolved into two components, one a high-energy form with nound NA ion and the other a low-energy form free of Na ion. An attempt will be made to detect these forms by 32P-NMR. With the purified enzyme solubilized in Triton protomer-tetramer equilibrium will be investigated by chromatography on Sepharose. Then the effects of the degree of polymerization on the phosphorylation kinetics will be investigated.